Method for purifying extracellular vesicles

A purification method and vesicle technology, which can be used in plant cells, isolate microorganisms, etc., and can solve problems such as protein contamination, unsatisfactory, cumbersome operations, etc.

Active Publication Date: 2021-09-14
INST OF URBAN ENVIRONMENT CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003]Existing extracellular vesicles are prone to protein contamination during the preparation process, which cannot meet the requirements of subsequent NTA (Nanoparticle Tracking Analysis) nanoparticle tracking analysis and electron microscope observation or proteome analysis, etc.
Extracellular vesicles of large volume samples are usually purified by gradient density method, but this method is costly, cumbersome and time-consuming

Method used

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  • Method for purifying extracellular vesicles
  • Method for purifying extracellular vesicles
  • Method for purifying extracellular vesicles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] The preparation of embodiment 1SEC column (size-exclusion chromatography column)

[0077] Wash Sepharose CL-2B (Sepharose) (Sepharose CL-2B Sepharose CL-2B, product number S8731-100mL) 3-4 times; 10mL disposable syringe, remove the needle, place a 20μm filter membrane (Mollipore ), placed vertically on the shelf. Slowly add the washed CL-2B into the syringe to ensure that the filtered liquid below is clear and transparent. Finally wash with 10mL PBS. SEC columns can be stored in a refrigerator at 4°C for 1 week.

Embodiment 2

[0078] Example 2 Purification of extracellular vesicles extracted by ultracentrifugation

[0079] A1, Escherichia coli E.Coil E4742 were cultured in 5L LB medium at 37°C and 150rpm / min until late exponential phase (OD600=1.2);

[0080] A2. Centrifuge the bacterial solution obtained in A1, centrifugation conditions: 4°C, 10000g, 15mins, to obtain the supernatant;

[0081] A3, the supernatant obtained in A2 is subjected to the first filtration treatment with a 0.45 μm filter membrane, and the filtrate is collected;

[0082] A4, the filtrate obtained in A3 is subjected to a second filtration treatment with a 0.22 μm filter membrane, and the filtrate is collected;

[0083] A5. Concentrate the filtrate obtained in A4 with a small tangential flow device (100kDa cutoff, Amicon, Merck) to finally obtain a 78mL concentrate;

[0084] A6. Pass the concentrated solution obtained in A5 through a 0.22 μm syringe filter (Merck Millipore, Ireland) for the third filtration treatment, and col...

Embodiment 3

[0096] Example 3 Purification of extracellular vesicles extracted by the kit (Bacterial Membrane Vesicle Isolation Kit (Bacterial Medium) 10ml, Runji Biology, Catnumber: BacMV10-10)

[0097] B1, Escherichia coli E.Coil E4742 were cultured in 1L LB medium at 37°C and 150rpm / min until late exponential phase (OD600=1.2);

[0098] B2. Centrifuge the bacterial liquid obtained in B1, centrifugation conditions: 4°C, 10000g, 15mins, to obtain the supernatant;

[0099] B3. The supernatant obtained in B2 is subjected to the first filtration treatment with a 0.45 μm filter membrane, and the filtrate is collected;

[0100] B4, the filtrate obtained in B3 is subjected to a second filtration treatment with a 0.22 μm filter membrane, and the filtrate is collected;

[0101] B5. Concentrate the filtrate obtained in B4 with a 100KD ultrafiltration tube (Merck Millipore, Ireland), centrifuge at 3000g at 4°C for 5-10mins, and finally obtain 20mL of the concentrate;

[0102] B6. Divide the conce...

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Abstract

The invention provides a method for purifying extracellular vesicles of a large-volume sample solution. The method comprises the following steps: providing a sample solution of extracellular vesicles to be prepared, and extracting the extracellular vesicles; taking the volume of the sample solution by liter; adding the extracellular vesicles into an exclusion chromatographic column, and collecting flow-down components in an isovolumetric manner; selecting and mixing the flow-down components having a target particle size or a target concentration of free protein; and transferring the mixture into an ultrafiltration tube for centrifugation, and reserving trapped fluid to obtain purified extracellular vesicles. The obtained extracellular vesicles are high in purity, small in soluble impure protein pollution and wide in practicability, and can fulfill subsequent analysis of NTA, electron microscopes, proteomes and the like. The operation is simple and costs short time.

Description

technical field [0001] The present disclosure relates to the field of biotechnology, in particular to a method for purifying extracellular vesicles. Background technique [0002] Extracellular vesicles (EVs) are a general term for various vesicle structures with membrane structures released by cells. Based on their biogenesis, size, and biophysical properties, they can be further classified (eg, exosomes, microvesicles, etc.). It is an important carrier of intercellular communication, disease diagnosis and prognostic circulating biomarkers, and has important application prospects. [0003] The existing extracellular vesicles are prone to protein contamination during the preparation process, which cannot meet the needs of subsequent NTA (Nanoparticle Tracking Analysis) nanoparticle tracking analysis, electron microscope observation or proteome analysis. For extracellular vesicles in large volume samples, the gradient density method is usually used for purification, but this...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/02C12N5/04
CPCC12N1/02C12N5/04
Inventor 黄乾生黄海宁郭子晗竹李婷
Owner INST OF URBAN ENVIRONMENT CHINESE ACAD OF SCI
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